Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in children, and is being recognized more in recent years as a significant cause of disease in the immunocompromised, and in exacerbations of chronic obstruction pulmonary disease in adults. Although hMPV was discovered nearly two decades ago, no vaccine or therapeutic is currently available for prevention or treatment of hMPV disease, and we have a poor understanding of protective immune responses generated in response to natural infection. The sole target of neutralizing antibodies is the hMPV fusion (F) protein. The overall objective of this research project grant is to determine the human humoral immune response to the hMPV F protein. The data generated from this research project addresses our overall hypothesis that potent neutralizing antibody activity against hMPV is a result of (1) extensive antibody somatic mutation, and of (2) recognition of specific epitopes on the F protein that inhibit structural transitions critical for the viral attachment and fusion processes. Utilizing blood samples from human subjects, human memory B cells will be screened for antibody-specificity targeting the pre- fusion and post-fusion forms of the hMPV F protein, at which point the B cells will be fused to myeloma cells to generate human hybridomas. Isolated human mAbs targeting the hMPV F protein will be characterized to determine affinity, fine epitope specificity, neutralization potency, and for select mAbs we will determine the three-dimensional structure in complex with the hMPV F protein. These data will inform rational vaccine design of an effective hMPV vaccine. Determining the major neutralizing epitopes on the hMPV F protein will inform vaccine design by identifying amino acid regions important for hMPV F immunity, which can be incorporated into subunit or scaffolded vaccines to elicit target antibodies. Additionally, we will examine if these mAbs can prevent hMPV disease in vivo, which will be a prelude to use in the clinic. In Specific Aim 1, we will determine the ontogeny, neutralizing mechanisms, subgroup-reactivity, and conformational specificity of several hMPV F- specific human mAbs in both adults and children. In Specific Aim 2, we will determine the structural correlates of potent antibody neutralization of hMPV. In Specific Aim 3, we will determine the therapeutic efficacy of our top candidate human mAbs for prevention and treatment of hMPV disease in both mice and cotton rat models of infection.